Bicyclic inhibitors of histone deacetylase

ABSTRACT

Provided are compounds of the formula 
                                                                               
and pharmaceutically acceptable salts and composition thereof, which are useful in the treatment of conditions associated with inhibition of HDAC (e.g. HDAC2).

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national stage filing ofInternational Application No. PCT/US2018/013262 filed Jan. 11, 2018,which in turn claims priority to U.S. Provisional Application No.62/445,022 filed Jan. 11, 2017 and U.S. Provisional Application No.62/555,298 filed Sep. 7, 2017, the entire contents of each of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Small BusinessInnovation Research (SBIR) grant 1R43AG048651-01A1 awarded by theNational Institute of Health (NIH). The government has certain rights inthe invention.

BACKGROUND

Inhibitors of histone deacetylases (HDAC) have been shown to modulatetranscription and to induce cell growth arrest, differentiation andapoptosis. HDAC inhibitors also enhance the cytotoxic effects oftherapeutic agents used in cancer treatment, including radiation andchemotherapeutic drugs. Marks, P., Rifkind, R. A., Richon, V. M.,Breslow, R., Miller, T., Kelly, W. K. Histone deacetylases and cancer:causes and therapies. Nat Rev Cancer, 1, 194-202, (2001); and Marks, P.A., Richon, V. M., Miller, T., Kelly, W. K. Histone deacetylaseinhibitors. Adv Cancer Res, 91, 137-168, (2004). Moreover, recentevidence indicates that transcriptional dysregulation may contribute tothe molecular pathogenesis of certain neurodegenerative disorders, suchas Huntington's disease, spinal muscular atrophy, amyotropic lateralsclerosis, and ischemia. Langley, B., Gensert, J. M., Beal, M. F.,Ratan, R. R. Remodeling chromatin and stress resistance in the centralnervous system: histone deacetylase inhibitors as novel and broadlyeffective neuroprotective agents. Curr Drug Targets CNS Neurol Disord,4, 41-50, (2005). A recent review has summarized the evidence thataberrant histone acetyltransferase (HAT) and histone deacetylases (HDAC)activity may represent a common underlying mechanism contributing toneurodegeneration. Moreover, using a mouse model of depression, Nestlerhas recently highlighted the therapeutic potential of histonedeacetylation inhibitors (HDAC5) in depression. Tsankova, N. M., Berton,O., Renthal, W., Kumar, A., Neve, R. L., Nestler, E. J. Sustainedhippocampal chromatin regulation in a mouse model of depression andantidepressant action. Nat Neurosci, 9, 519-525, (2006).

There are 18 known human histone deacetylases, grouped into four classesbased on the structure of their accessory domains. Class I includesHDAC1, HDAC2, HDAC3, and HDAC8 and has homology to yeast Rpd3. HDAC4,HDAC5, HDAC7, and HDAC9 belong to class Ha and have homology to yeastHda1. HDAC6 and HDAC10 contain two catalytic sites and are classified asclass IIb. Class III (the sirtuins) includes SIRT1, SIRT2, SIRT3, SIRT4,SIRT5, SIRT6, and SIRT7. HDAC11 is another recently identified member ofthe HDAC family and has conserved residues in its catalytic center thatare shared by both class I and class II deacetylases and is sometimesplaced in class IV.

HDACs have been shown to be powerful negative regulators of long-termmemory processes. Nonspecific HDAC inhibitors enhance synapticplasticity as well as long-term memory (Levenson et al., 2004, J. Biol.Chem. 279:40545-40559; Lattal et al., 2007, Behav Neurosci121:1125-1131; Vecsey et al., 2007, J. Neurosci 27:6128; Bredy, 2008,Learn Mem 15:460-467; Guan et al., 2009, Nature 459:55-60; Malvaez etal., 2010, Biol. Psychiatry 67:36-43; Roozendaal et al., 2010, J.Neurosci. 30:5037-5046). For example, HDAC inhibition can transform alearning event that does not lead to long-term memory into a learningevent that does result in significant long-term memory (Stefanko et al.,2009, Proc. Natl. Acad. Sci. USA 106:9447-9452). Furthermore, HDACinhibition can also generate a form of long-term memory that persistsbeyond the point at which normal memory fails. HDAC inhibitors have beenshown to ameliorate cognitive deficits in genetic models of Alzheimer'sdisease (Fischer et al., 2007, Nature 447:178-182; Kilgore et al., 2010,Neuropsychopharmacology 35:870-880). These demonstrations suggest thatmodulating memory via HDAC inhibition have considerable therapeuticpotential for many memory and cognitive disorders.

The role of individual HDACs in long-term memory has been explored intwo recent studies. Kilgore et al. 2010, Neuropsychopharmacology35:870-880 revealed that nonspecific HDAC inhibitors, such as sodiumbutyrate, inhibit class I HDACs (HDAC1, HDAC2, HDAC3, HDAC8) with littleeffect on the class IIa HDAC family members (HDAC4, HDAC5, HDAC7,HDAC9). This suggests that inhibition of class I HDACs may be criticalfor the enhancement of cognition observed in many studies. Indeed,forebrain and neuron specific overexpression of HDAC2, but not HDAC1,decreased dendritic spine density, synaptic density, synaptic plasticityand memory formation. (Guan et al., 2009, Nature, 459:55-60). Incontrast, HDAC2 knockout mice exhibited increased synaptic density,increased synaptic plasticity and increased dendritic density inneurons. These HDAC2 deficient mice also exhibited enhanced learning andmemory in a battery of learning behavioral paradigms. This workdemonstrates that HDAC2 is a key regulator of synaptogenesis andsynaptic plasticity. Additionally, Guan et al. showed that chronictreatment of mice with SAHA (an HDAC 1, 2, 3, 6, 8 inhibitor) reproducedthe effects seen in the HDAC2 deficient mice and rescued the cognitiveimpairment in the HDAC2 overexpressing mice.

The inhibition of HDAC2 (selectively or in combination with inhibitionof other class I HDACs; as the primary target, or as part of a complexwith other proteins) is an attractive therapeutic target. Selectiveinhibition might be achieved by targeting specific HDAC isoforms such asHDAC2, in isolation, or as part of a functional multi-protein complex.Such inhibition has the potential for enhancing cognition andfacilitating the learning process through increasing synaptic anddendritic density in neuronal cell populations. In addition, inhibitionof specific HDACs, such as HDAC2, may also be therapeutically useful intreating a wide variety of other diseases and disorders.

SUMMARY

Disclosed are compounds and pharmaceutically acceptable salts thereof,and pharmaceutical compositions, which are useful in the treatment ofconditions associated with the activity of HDAC (e.g., HDAC2). (Seee.g., Table 2).

One of the advantages of certain compounds described herein is that theypossess improved brain exposure and have higher free brainconcentration. For example, the brain Cmax exposure and projected freebrain concentration of Compound 14 is 4-fold higher than comparator 1(which only differs from Compound 14 by the absence of a methyl group.See Table 5) when comparator 1 data is scaled for comparison. Inaddition, Compound 17 (which only differs by the presence of an ethylgroup when compared with comparator 1) is nearly 7-fold higher in brainCmax exposure and 5-fold higher in projected free brain concentrationwhen comparator 1 data is scaled for comparison. See Table 5.

Certain compounds described herein provide enhanced safety parameters.For example, replacement of the bottom phenyl ring for heteraromaticsafforded compounds with improved performance in colony forming unitassays, showing fewer effects on human erythroid and myeloidprogenitors. See e.g., Table 4, Compounds 4, 8, 11, and 12 compared tocomparator 2 and 3. Changing a 4-fluorophenyl group for a2,4-difluorophenyl group also has a profound effect in safetyparameters. See Table 4, Comparator 6 with Compound 9.

Certain compounds described herein perform well in a cell lysate assayemploying an exogenous substrate to measure HDAC inhibitory activity.See Table 3.

Conditions which are treatable by the disclosed compounds include, butare not limited to, neurological disorders, memory or cognitive functiondisorders or impairments, extinction learning disorders, fungal diseasesor infections, inflammatory diseases, hematological diseases, neoplasticdiseases, psychiatric disorders, and memory loss.

DETAILED DESCRIPTION 1. Compounds

Provided herein are compounds of the formula:

or a pharmaceutically acceptable salt thereof.

2. Definitions

As used herein the terms “subject” and “patient” may be usedinterchangeably, and means a mammal in need of treatment, e.g.,companion animals (e.g., dogs, cats, and the like), farm animals (e.g.,cows, pigs, horses, sheep, goats and the like) and laboratory animals(e.g., rats, mice, guinea pigs and the like). Typically, the subject isa human in need of treatment.

Pharmaceutically acceptable salts as well as the neutral forms of thecompounds described herein are included. For use in medicines, the saltsof the compounds refer to non-toxic “pharmaceutically acceptable salts.”Pharmaceutically acceptable salt forms include pharmaceuticallyacceptable acidic/anionic or basic/cationic salts. Pharmaceuticallyacceptable basic/cationic salts include, the sodium, potassium, calcium,magnesium, diethanolamine, n-methyl-D-glucamine, L-lysine, L-arginine,ammonium, ethanolamine, piperazine and triethanolamine salts.Pharmaceutically acceptable acidic/anionic salts include, e.g., theacetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, carbonate,citrate, dihydrochloride, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrobromide, hydrochloride, malate, maleate,malonate, mesylate, nitrate, salicylate, stearate, succinate, sulfate,tartrate, and tosylate.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier, adjuvant, or vehicle that does not destroy the pharmacologicalactivity of the compound with which it is formulated. Pharmaceuticallyacceptable carriers, adjuvants or vehicles that may be used in thecompositions described herein include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, reducing the likelihood of developing, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed, i.e., therapeutic treatment.In other embodiments, treatment may be administered in the absence ofsymptoms. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors),i.e., prophylactic treatment. Treatment may also be continued aftersymptoms have resolved, for example to prevent or delay theirrecurrence.

The term “effective amount” or “therapeutically effective amount”includes an amount of a compound described herein that will elicit abiological or medical response of a subject.

3. Uses, Formulation and Administration

In some embodiments, compounds and compositions described herein areuseful in treating conditions associated with the activity of HDAC. Suchconditions include for example, those described below.

Recent reports have detailed the importance of histone acetylation incentral nervous system (“CNS”) functions such as neuronaldifferentiation, memory formation, drug addiction, and depression(Citrome, Psychopharmacol. Bull. 2003, 37, Suppl. 2, 74-88; Johannessen,CNS Drug Rev. 2003, 9, 199-216; Tsankova et al., 2006, Nat. Neurosci. 9,519-525; Bousiges et al., 2013, PLoS ONE 8(3), e57816). Thus, in oneaspect, the provided compounds and compositions may be useful intreating a neurological disorder. Examples of neurological disordersinclude: (i) chronic neurodegenerative diseases such as fronto-temporallobar degeneration (frontotemporal dementia, FTD), FTD-GRN, familial andsporadic amyotrophic lateral sclerosis (FALS and ALS, respectively),familial and sporadic Parkinson's disease, Huntington's disease,familial and sporadic Alzheimer's disease, multiple sclerosis, musculardystrophy, olivopontocerebellar atrophy, multiple system atrophy,Wilson's disease, progressive supranuclear palsy, diffuse Lewy bodydisease, corticodentatonigral degeneration, progressive familialmyoclonic epilepsy, striatonigral degeneration, torsion dystonia,familial tremor, Down's Syndrome, Gilles de la Tourette syndrome,Hallervorden-Spatz disease, diabetic peripheral neuropathy, dementiapugilistica, AIDS Dementia, age related dementia, age associated memoryimpairment, and amyloidosis-related neurodegenerative diseases such asthose caused by the prion protein (PrP) which is associated withtransmissible spongiform encephalopathy (Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, scrapie, and kuru), and thosecaused by excess cystatin C accumulation (hereditary cystatin Cangiopathy); and (ii) acute neurodegenerative disorders such astraumatic brain injury (e.g., surgery-related brain injury), cerebraledema, peripheral nerve damage, spinal cord injury, Leigh's disease,Guillain-Barre syndrome, lysosomal storage disorders such aslipofuscinosis, Alper's disease, restless leg syndrome, vertigo asresult of CNS degeneration; pathologies arising with chronic alcohol ordrug abuse including, for example, the degeneration of neurons in locuscoeruleus and cerebellum, drug-induced movement disorders; pathologiesarising with aging including degeneration of cerebellar neurons andcortical neurons leading to cognitive and motor impairments; andpathologies arising with chronic amphetamine abuse to includingdegeneration of basal ganglia neurons leading to motor impairments;pathological changes resulting from focal trauma such as stroke, focalischemia, vascular insufficiency, hypoxic-ischemic encephalopathy,hyperglycemia, hypoglycemia or direct trauma; pathologies arising as anegative side-effect of therapeutic drugs and treatments (e.g.,degeneration of cingulate and entorhinal cortex neurons in response toanticonvulsant doses of antagonists of the NMDA class of glutamatereceptor) and Wernicke-Korsakoff's related dementia. Neurologicaldisorders affecting sensory neurons include Friedreich's ataxia,diabetes, peripheral neuropathy, and retinal neuronal degeneration.Other neurological disorders include nerve injury or trauma associatedwith spinal cord injury. Neurological disorders of limbic and corticalsystems include cerebral amyloidosis, Pick's atrophy, and Rett syndrome.In another aspect, neurological disorders include disorders of mood,such as affective disorders and anxiety; disorders of social behavior,such as character defects and personality disorders; disorders oflearning, memory, and intelligence, such as mental retardation anddementia. Thus, in one aspect the disclosed compounds and compositionsmay be useful in treating schizophrenia, delirium, attention deficithyperactivity disorder (ADHD), schizoaffective disorder, Alzheimer'sdisease, vascular dementia, Rubinstein-Taybi syndrome, depression,mania, attention deficit disorders, drug addiction, dementia, dementiaincluding BPSD manifestations, agitation, apathy, anxiety, psychoses,personality disorders, bipolar disorders, unipolar affective disorder,obsessive-compulsive disorders, eating disorders, post-traumatic stressdisorders, irritability, adolescent conduct disorder and disinhibition.They may also be useful for spontaneous, toxic, neoplastic,post-traumatic and post-infectious tinnitus and smelling impairment.

Transcription is thought to be a key step for long-term memory formation(Alberini, 2009, Physiol. Rev. 89, 121-145). Transcription is promotedby specific chromatin modifications, such as histone acetylation, whichmodulate histone-DNA interactions (Kouzarides, 2007, Cell, 128:693-705),as well as transcription factor-DNA interactions. Modifying enzymes,such as histone acetyltransferases (HATs) and histone deacetylases(HDACs), regulate the state of acetylation on histone tails. In general,histone acetylation promotes gene expression, whereas histonedeacetylation leads to gene silencing, although treatment with HDACinhibitors can result in both upregulation and downregulation of theexpression levels of specific genes. Numerous studies have shown that apotent HAT, cAMP response element-binding protein (CREB)-binding protein(CBP), is necessary for long-lasting forms of synaptic plasticity andlong term memory (for review, see Barrett, 2008, Learn Mem 15:460-467).Thus, in one aspect, the provided compounds and compositions may beuseful for promoting cognitive function and enhancing learning andmemory formation.

The compounds and compositions described herein may also be used fortreating fungal diseases or infections.

In another aspect, the compounds and compositions described herein maybe used for treating inflammatory diseases such as stroke, rheumatoidarthritis, lupus erythematosus, ulcerative colitis and traumatic braininjuries (Leoni et al., PNAS, 99(5); 2995-3000(2002); Suuronen et al. J.Neurochem. 87; 407-416 (2003) and Drug Discovery Today, 10: 197-204(2005).

In yet another aspect, the compounds and compositions described hereinmay be used for treating a cancer caused by the proliferation ofneoplastic cells. Such cancers include e.g., solid tumors, neoplasms,carcinomas, sarcomas, leukemias, lymphomas and the like. In one aspect,cancers that may be treated by the compounds and compositions describedherein include, but are not limited to: cardiac cancer, lung cancer,gastrointestinal cancer, genitourinary tract cancer, liver cancer,nervous system cancer, gynecological cancer, hematologic cancer, skincancer, and adrenal gland cancer. In one aspect, the compounds andcompositions described herein are useful in treating cardiac cancersselected from sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma. Inanother aspect, the compounds and compositions described herein areuseful in treating a lung cancer selected from bronchogenic carcinoma(squamous cell, undifferentiated small cell, undifferentiated largecell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, and mesothelioma.In one aspect, the compounds and compositions described herein areuseful in treating a gastrointestinal cancer selected from esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors,Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma),and large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma). In one aspect, the compounds and compositionsdescribed herein are useful in treating a genitourinary tract cancerselected from kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma,sarcoma), and testis (seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma,fibroma, fibroadenoma, adenomatoid tumors, lipoma). In one aspect, thecompounds and compositions described herein are useful in treating aliver cancer selected from hepatoma (hepatocellular carcinoma),cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellularadenoma, and hemangioma.

In some embodiments, the compounds described herein relate to treating,a bone cancer selected from osteogenic sarcoma (osteosarcoma),fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing'ssarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma,malignant giant cell tumor chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors.

In one aspect, the compounds and compositions described herein areuseful in treating a nervous system cancer selected from skull (osteoma,hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastomamultiform, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), and spinal cord (neurofibroma, meningioma, glioma, sarcoma).

In one aspect, the compounds and compositions described herein areuseful in treating a gynecological cancer selected from uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),and fallopian tubes (carcinoma).

In one aspect, the compounds and compositions described herein areuseful in treating a skin cancer selected from malignant melanoma, basalcell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, andpsoriasis.

In one aspect, the compounds and compositions described herein areuseful in treating an adrenal gland cancer selected from neuroblastoma.

In one aspect, the compounds and compositions described herein areuseful in treating cancers that include, but are not limited to:leukemias including acute leukemias and chronic leukemias such as acutelymphocytic leukemia (ALL), Acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and HairyCell Leukemia; lymphomas such as cutaneous T-cell lymphomas (CTCL),noncutaneous peripheral T-cell lymphomas, lymphomas associated withhuman T-cell lymphotrophic virus (HTLV) such as adult T-cellleukemia/lymphoma (ATLL), Hodgkin's disease and non-Hodgkin's lymphomas,large-cell lymphomas, diffuse large B-cell lymphoma (DLBCL); Burkitt'slymphoma; mesothelioma, primary central nervous system (CNS) lymphoma;multiple myeloma; childhood solid tumors such as brain tumors,neuroblastoma, retinoblastoma, Wilm's tumor, bone tumors, andsoft-tissue sarcomas, common solid tumors of adults such as head andneck cancers (e.g., oral, laryngeal and esophageal), genito urinarycancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular,rectal and colon), lung cancer, breast cancer, pancreatic cancer,melanoma and other skin cancers, stomach cancer, brain tumors, livercancer and thyroid cancer.

In one aspect, the compounds and compositions described herein areuseful in treating a condition in a subject selected from a neurologicaldisorder, memory or cognitive function disorder or impairment,extinction learning disorder, fungal disease or infection, inflammatorydisease, hematological disease, psychiatric disorders, and neoplasticdisease. In another aspect, the compounds and compositions describedherein are useful in treating a condition selected from a) a cognitivefunction disorder or impairment associated with Alzheimer's disease,Huntington's disease, seizure induced memory loss, schizophrenia,Rubinstein Taybi syndrome, Rett Syndrome, Fragile X, Lewy body dementia,vascular dementia, fronto-temporal lobar degeneration (frontotemporaldementia, FTD), FTD-GRN, ADHD, dyslexia, bipolar disorder and social,cognitive and learning disorders associated with autism, traumatic headinjury, attention deficit disorder, anxiety disorder, conditioned fearresponse, panic disorder, obsessive compulsive disorder, posttraumaticstress disorder (PTSD), phobia, social anxiety disorder, substancedependence recovery, Age Associated Memory Impairment (AAMI), AgeRelated Cognitive Decline (ARCD), ataxia, or Parkinson's disease; b) ahematological disease selected from acute myeloid leukemia, acutepromyelocytic leukemia, acute lymphoblastic leukemia, chronicmyelogenous leukemia, myelodysplastic syndromes, and sickle cell anemia;c) a neoplastic disease; and d) an extinction learning disorder selectedfrom fear extinction and post-traumatic stress disorder. In one aspect,the condition treated by the compounds and compostions described hereinis the condition is Alzheimer's disease, Huntington's disease,frontotemporal dementia, Freidreich's ataxia, post-traumatic stressdisorder (PTSD), Parkinson's disease, depression, or substancedependence recovery.

In one aspect, the present disclosure provides a method of treating acondition described herein comprising administering to a subject aneffective amount of a compound, or pharmaceutically acceptable saltdescribed herein, or a composition thereof.

Also provided is the use of one or more of the compounds, orpharmaceutically acceptable salts thereof described herein, or aprovided composition, for treating a condition described herein.

Also provided is the use of one or more of the compounds, orpharmaceutically acceptable salts thereof described herein for themanufacture of a medicament for treating a condition described herein.

Subjects may also be selected to be suffering from one or more of thedescribed conditions before treatment with one or more of the describedcompounds, or pharmaceutically acceptable salts or compositionscommences.

The present disclosure also provides pharmaceutically acceptablecompositions comprising a compound described herein, or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier. These compositions can be used to treat one or moreof the conditions described above.

Compositions described herein may be administered orally, parenterally,by inhalation spray, topically, rectally, nasally, buccally, vaginallyor via an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. Liquid dosage forms,injectable preparations, solid dispersion forms, and dosage forms fortopical or transdermal administration of a compound are included herein.

The amount of provided compounds that may be combined with carriermaterials to produce a composition in a single dosage form will varydepending upon the patient to be treated and the particular mode ofadministration. In some embodiments, provided compositions may beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe provided compound, such as e.g., 0.1-100 mg/kg body weight/day, canbe administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including age, body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, the judgment of thetreating physician, and the severity of the particular disease beingtreated. The amount of a provided compound in the composition will alsodepend upon the particular compound in the composition.

EXEMPLIFICATION

Spots were visualized by UV light (254 and 365 nm). Purification bycolumn and flash chromatography was carried out using silica gel(200-300 mesh). Solvent systems are reported as the ratio of solvents.

NMR spectra were recorded on a Bruker 400 (400 MHz) spectrometer. ¹Hchemical shifts are reported in 6 values in ppm with tetramethylsilane(TMS, =0.00 ppm) as the internal standard. See, e.g., the data providedin Table 1.

LCMS spectra were obtained on an Agilent 1200 series 6110 or 6120 massspectrometer with ESI (+) ionization mode. See, e.g., the data providedin Table 1.

Example 1

Synthesis of SM-A.

A mixture of 6-chloro-3-nitropyridin-2-amine (10.0 g, 57.6 mmol),4-fluorophenylboronic acid (8.87 g, 63.4 mmol) and Cs₂CO₃ (37.56 g,115.2 mmol) in dioxane/H₂O (200 mL/20 mL) was treated with Pd(PPh₃)₄(2.44 g, 2.9 mmol) under a N₂ atmosphere. The mixture was stirred at 95°C. for 2 h and then concentrated in vacuo. The residue was dissolvedwith EtOAc (200 mL) and the solution was washed with brine (100 mL×3).The organic layer was dried over anhydrous Na₂SO₄ and then concentratedin vacuo. The residue was purified by column chromatography on silicagel (PE:EtOAc=10:1˜3:1) to give SM-A (11.2 g, 83%) as a yellow solid. MS234.2 [M+H]⁺.

Synthesis of SM-B.

To a stirred solution of SM-A (3.0 g, 13.0 mmol) in pyridine (60 mL) wasadded phenyl carbonochloridate (4.45 g, 28.5 mmol) dropwise while thereaction mixture was cooled with an ice bath. The resulting mixture wasthen warmed to RT following addition, and then was stirred at 50° C. for4 h. The mixture was concentrated in vacuo, and the residue was purifiedby column chromatography on silica gel (PE:EtOAc=8:1˜3:1) to give SM-B(5.2 g, 84%) as a yellow solid. MS 474.4 [M+H]⁺.

Synthesis of 1641-A and 1641-A1.

To a solution of tert-butyl 4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate (450 mg, 2.15 mmol) in DMF (8 mL) was addedNaH (60% in mineral oil) (155 mg, 3.87 mmol) under ice bath cooling, andthe reaction mixture was than allowed to warm to room temperature andstirred at room temperature for 30 min. 3-Bromooxetane (471 mg, 3.44mmol) was then added to the reaction mixture, and the reaction wasstirred at 70° C. overnight. The mixture was quenched with ice water (50mL), extracted with EtOAc (15 mL×3), and the combined organic layerswere washed with brine (12 mL×3), dried over anhydrous Na₂SO₄ and thenconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (PE:EtOAc=3:1˜1:1) to give 1641-A and 1641-A1 (410 mg,72%) as a colorless oil. MS 266.0 [M+H]⁺.

Synthesis of 1641-B and 1642-B1.

To a solution of 1641-A and 1642-A1 (410 mg, 1.55 mmol) in DCM (12 mL)was added TFA (3 mL), and the reaction mixture was stirred at roomtemperature for 1 h. The solvent was removed in vacuo to give 1641-B and1641-B1 as a crude product which was carried on to the next step withoutfurther purification. MS 166.1 [M+H]⁺.

Synthesis of 1641-C and 1641-C1.

To a solution of 1641-B and 1641-B1 (crude product from last step) andSM-B (407 mg, 0.86 mmol) in MeCN (15 mL) was added Na₂CO₃ (912 mg, 8.6mmol). The resulting reaction mixture was stirred at 50° C. for 2 h,whereupon the mixture was filtered and the filtrate was concentrated invacuo. The residue was purified by column chromatography on silica gel(DCM:MeOH=80:1˜50:1) to give 1641-C and 1641-C1 (230 mg, 63%) as ayellow solid. MS 425.0 [M+H]⁺.

Synthesis of Compound 1 and T-1641A.

A mixture of 1641-C and 1641-C1 (230 mg, 0.54 mmol) and Pd/C (200 mg) inMeOH (30 mL) was stirred under a H₂ atmosphere (1 atm) at roomtemperature for 1 h. Pd/C was then removed by filtration through theCelite, and the filtrate was concentrated. The crude residue waspurified by HPLC using chiral separation (Column: Chiralcel OD-3;Solvent: MeOH; Flow rate: 2 mL/min; RT₁₆₄₁=2.359 min, RT_(1641A)=3.066min) to give Compound 1 (110 mg, 52%) as a white solid (MS 395.2 [M+H]⁺)and T-1641A (55 mg, 26%) as a white solid. MS 395.2 [M+H]⁺.

Example 2

Synthesis of 1677-A.

To a solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (2.0 g, 10.8mmol) in THF (20 mL) was added DMF-DMA (3.8 g, 32.4 mmol). The reactionmixture was stirred at 70° C. for 12 h and then concentrated to give1677-A (4.0 g) as a crude product which was carried on without furtherpurification. MS 241.2 [M+H]⁺.

Synthesis of 1677-B.

To a solution of 1677-A (2.0 g, crude product from last step) in EtOH(20 mL) was added acetimidamide hydrochloride (3.1 g, 33.0 mmol) andEt₃N (3.3 g, 33.0 mmol). The reaction mixture was stirred at 80° C. for16 h, then was diluted with water (50 mL), and extracted with EtOAc (20mL×3). The combined organic layers were washed with brine (20 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel (PE:EtOAc=0:1˜10:1)to give 1677-B (1.6 g, 83%) as a brown solid. MS 236.1 [M+H]⁺.

Synthesis of 1677-C.

To a solution of 1677-B (800 mg, 3.4 mmol) in DCM (6 mL) was added TFA(6 mL) dropwise while cooling the reaction mixture in an ice bath. Thereaction mixture was allowed to warm to room temperature, and was thenstirred at room temperature for 1 h. The solvent was removed in vacuo togive 1677-C (850 mg) as a crude product which was carried on withoutfurther purification. MS 136.0 [M+H]⁺.

Synthesis of 1677-D.

A mixture of 6-chloro-3-nitropyridin-2-amine (670 mg, 3.75 mmol),2-(5-fluorothiophen-2-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (898mg, 3.94 mmol) and K₂CO₃ (1.55 g, 11.25 mmol) in dioxane/H₂O (25 mL/2.5mL) was treated with Pd(PPh₃)₄ (216 mg, 0.19 mmol) under a N₂atmosphere. The reaction mixture was stirred at 95° C. for 3 h and thenconcentrated in vacuo. The crude residue was dissolved with EtOAc (200mL) and the resulting solution was washed with brine (100 mL×3). Theorganic layer was dried over anhydrous Na₂SO₄ and then concentrated invacuo. The residue was purified by column chromatography on silica gel(PE:EtOAc=7:1˜5:1) to give 1677-D (900 mg, 93%) as a yellow solid. MS240.1 [M+H]⁺.

Synthesis of 1677-E.

To a stirred solution of 1677-D (460 mg, 1.92 mmol) in pyridine (10 mL)was added phenyl carbonochloridate (900 mg, 5.77 mmol) dropwise. Afterthe addition was completed, the mixture was stirred at 55° C. for 2 h.The mixture was concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (PE:DCM=20:1˜1:3) to give 1677-E(700 mg, 76%) as a yellow solid. MS 479.8 [M+H]⁺.

Synthesis of 1677-F.

To a solution of 1677-E (200 mg, 0.42 mmol) and 1677-C (116 mg, crudeproduct) in DMSO (5 mL) was added NaHCO₃ (352 mg, 4.2 mmol). Thereaction mixture was stirred at room temperature for 2 h, whereupon themixture was diluted with water (50 mL), and extracted with EtOAc (20mL×3). The combined organic layers were washed with brine (20 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by Prep-TLC (EA:PE=5:1) to give 1677-F (80 mg, 48%) as abrown solid. MS 400.9 [M+H]⁺.

Synthesis of Compound 2.

A mixture of 1677-F (68 mg, 0.17 mmol) and Raney Ni (20 mg) in DCM/MeOH(3 mL/5 mL) was stirred at room temperature for 0.5 h. The Ni was thenremoved by filtration through Celite, the filtrate was concentrated, andthe crude residue was purified by Prep-TLC (DCM:MeOH=10:1) to giveCompound 2 (20 mg, 27%) as a gray solid. MS 371.2 [M+H]⁺.

Example 3

Synthesis of SM-H.

A mixture of 6-chloro-3-nitropyridin-2-amine (10.00 g, 57.6 mmol),thiophen-2-ylboronic acid (8.12 g, 63.4 mmol) and Cs₂CO₃ (37.56 g, 115.2mmol) in dioxane/H₂O (200 mL/20 mL) was treated with Pd(PPh₃)₄ (2.44 g,2.88 mmol) under a N₂ atmosphere. The reaction mixture was stirred at95° C. for 2 h and then concentrated in vacuo. The crude residue wasdissolved with EtOAc (200 mL) and the resulting solution was washed withbrine (100 mL×3). The organic layer was dried over anhydrous Na₂SO₄ andthen concentrated in vacuo. The residue was purified by columnchromatography on silica gel (PE:EtOAc=5:1˜3:1) to give SM-H (10.0 g,79%) as a yellow solid. MS 222.2 [M+H]⁺.

Synthesis of SM-J.

To a solution of SM-H (1.30 g, 5.88 mmol) in pyridine (20 mL) was addedphenyl carbonochloridate (2.29 g, 14.7 mmol) dropwise. After theaddition was completed, the mixture was heated to 50° C. and stirred atthat temperature for 4 h. The mixture was then concentrated in vacuo,and the crude residue was purified by column chromatography on silicagel (PE:EtOAc=8:1˜3:1) to give SM-J (2.4 g, 89%) as a yellow solid. MS462.4 [M+H]⁺.

Synthesis of 1687-A.

A solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (4.0 g, 21.6mmol) and DMF-DMA (7.6 g, 64.8 mmol in THF (40 mL) was stirred at 70° C.for 16 h. The solution was concentrated in vacuo to give 1687-A as acrude product which was used directly in the next step withoutpurification. MS 241.3 [M+H]⁺.

Synthesis of 1687-B.

To a solution of 1687-A (8.2 mmol, crude product from last step) in EtOH(10 mL) was added Et₃N (4.1 g, 41.0 mmol) and propionimidamidehydrochloride (3.55 g, 32.8 mmol). The resulting reaction mixture wasstirred at 80° C. for 24 h. After the mixture was cooled to roomtemperature, the mixture was diluted with water (50 mL) and extractedwith DCM (30 mL×3). The combined organic layers were washed with brine(30 mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo.The crude residue was purified by column chromatography on silica gel(PE:DCM=10:1˜1:2) to give 1687-B (1.0 g, 49%) as a brown solid. MS 250.3[M+H]⁺.

Synthesis of 1687-C.

To a solution of 1687-B (1.0 g, 4.02 mmol) in DCM (10 mL) was added TFA(5 mL) dropwise. The reaction mixture was stirred at room temperaturefor 1 h, and then the solution was concentrated in vacuo to give 1687-Cas a crude product which was used directly in the next step withoutpurification. MS 150.3 [M+H]⁺.

Synthesis of 1687-D.

A mixture of SM-J (292 mg, 0.63 mmol) and 1687-C (0.82 mmol) in DMSO (8mL) was treated with Na₂CO₃ (537 mg, 5.07 mmol), and the resultingreaction mixture was stirred at room temperature for 2 h. The mixturewas then diluted with water (20 mL), extracted with EtOAc (20 mL×3), andthe combined organic layers were washed with brine (20 mL×3), dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The crude residue waspurified by column chromatography on silica gel (DCM:MeOH=100:1˜50:1) togive 1687-D (150 mg, 60%) as a yellow solid. MS 397.4 [M+H]⁺.

Synthesis of Compound 3.

A mixture of 1687-D (100 mg, 0.25 mmol) and Pd/C (100 mg) in MeOH (10mL) was stirred under a H₂ atmosphere (1 atm) at room temperature for 1h. The Pd/C was then removed by filtration through Celite, the filtratewas concentrated, and the crude residue was purified by Prep-TLC(DCM:MeOH=10:1) to give Compound 3 (45 mg, 49%) as a yellow solid (MS367.4 [M+H]⁺).

Example 4

Synthesis of SM-E.

To a solution of 6-bromo-3-nitropyridin-2-amine (5.0 g, 23.0 mmol) andEt₃N (6.9 g, 69.0 mmol) in THF (60 mL) cooled to 0° C. was added phenylcarbonochloridate (10.8 g, 69.0 mmol) dropwise. After the addition wascompleted, the mixture was allowed to warm to room temperature, and wasstirred at room temperature for 1 h. After 1 h, the reaction mixture wasfiltered and concentrated in vacuo. The residue was recrystallized frompetroleum ether to give SM-E (10.2 g, 97%) as a light yellow solid. MS458.0, 460.0 [M+H]⁺.

Synthesis of 1701-A and 1701-A1.

To a solution of tert-butyl 4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate (1.00 g, 4.78 mmol) in DMF (10 mL) cooled to0° C. was added NaH (60% in mineral oil) (420 mg, 10.5 mmol), and theresulting reaction mixture was allowed to warm to room temperature andstirred at room temperature for 30 min. At this point, MeI (814 mg, 5.74mmol) was added into the reaction mixture and stirring at roomtemperature was continued for 2 h. The reaction was then quenched withwater (50 mL), extracted with EtOAc (30 mL×3), and the combined organiclayers were washed with brine (10 mL×3), dried over anhydrous Na₂SO₄ andthen concentrated in vacuo to give crude 1701-A and 1701-A1 (920 mg,86%) as a yellow oil. The crude product was taken on to the next stepwithout further purification. MS 224.1 [M+H]⁺.

Synthesis of 1701-B and 1701-B1.

To a round bottomed flask containing a mixture of 1701-A and 1701-A1(920 mg, 4.13 mmol) and cooled with an ice bath was addedHCl/1,4-dioxane (4N, 10 mL). The reaction mixture was stirred at roomtemperature for 1 h. The solvent was then removed in vacuo to give1701-B and 1701-B1 as a crude product which was taken on to the nextstep without purification. MS 124.1 [M+H]⁺.

Synthesis of 1701-C.

To a solution of 1701-B and 1701-B1 (crude product from last step) andSM-E (800 mg, 1.75 mmol) in DMSO (10 mL) was added Na₂CO₃ (1.48 g, 13.97mmol). The mixture was stirred at room temperature for 4 h. The mixturewas diluted with water (50 mL), extracted with EtOAc (20 mL×3). Thecombined organic layer was washed with brine (20 mL×3), dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The residue waspurified by column chromatography on silica gel (DCM:MeOH=100:1˜30:1) togive 1701-C and 1701-C1 as a yellow solid. The regioisomers were thenseparated by Prep-TLC (DCM:EA=4:1) to give 1701-C (150 mg, 23%) as ayellow solid. MS 367.0, 369.0 [M+H]⁺.

Synthesis of 1701-D.

A mixture of 1701-C (150 mg, 0.41 mmol),2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole (120mg, 0.53 mmol) and K₂CO₃ (169 mg, 1.23 mmol) in dioxane/H₂O (10 mL/2 mL)was treated with Pd(PPh₃)₄ (23.6 mg, 0.02 mmol) under a N₂ atmosphere.The reaction mixture was stirred at 50° C. for 16 h, and was thenconcentrated in vacuo. The crude residue was dissolved with EtOAc (20mL), and the resulting solution was washed with brine (10 mL×3). Theorganic layer was dried over anhydrous Na₂SO₄ and then concentrated invacuo. The crude residue was purified by Prep-TLC (DCM:MeOH=20:1) togive 1701-D (90 mg, 57%) as a yellow solid. MS 386.4 [M+H]⁺.

Synthesis of Compound 4.

A mixture of 1701-D (90 mg, 0.23 mmol) and Raney Ni (90 mg) in MeOH/DCM(10 mL/2 mL) was stirred under a H₂ atmosphere (1 atm) at roomtemperature for 1 h. The Raney Ni was then removed by filtration throughCelite, the filtrate was concentrated, and the crude residue waspurified by Prep-TLC (DCM:MeOH=10:1) to give Compound 4 (25 mg, 31%) asa gray solid (MS 356.4 [M+H]⁺).

Example 5

Synthesis of 1783-A and 1783-A1.

A solution of tert-butyl 4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate (600 mg, 2.88 mmol) in DMF (8 mL) was cooledto 0° C., and then was treated with NaH (60% in mineral oil) (184 mg,4.6 mmol). The reaction was allowed to warm to room temperature, wasstirred at room temperature for 30 min, and then1,1-difluoro-2-iodoethane (829 mg, 4.32 mmol) was added. After stirringat room temperature for 3 h, the mixture was quenched with water (45 mL)and extracted with EtOAc (14 mL×3). The combined organic layers werewashed with brine (10 mL×3), dried over anhydrous Na₂SO₄ and thenconcentrated in vacuo. The crude residue was purified by columnchromatography on silica gel (PE:EtOAc=3:1˜3:2) to give a mixture of1783-A and 1783-A1 (520 mg, 66%) as a white solid. MS 274.1 [M+H]⁺.

Synthesis of 1783-B and 1783-B1.

A mixture of 1783-A and 1783-A1 (520 mg, 1.9 mmol) was treated withHCl/EtOAc (4 M, 16 mL), and the reaction mixture was stirred at roomtemperature for 1 h. The solvent was then removed in vacuo to give amixture of 1783-B and 1783-B1 (460 mg, 98%) as gray solid which wastaken on to the next step without purification. MS 174.0 [M+H]⁺.

Synthesis of 1783-C and 1783-C1.

A solution of the regioisomeric mixture of 1783-B and 1783-B1 (460 mg,1.87 mmol) and SM-E (450 mg, 0.98 mmol) in DMSO (10 mL) was treated withNa₂CO₃ (1.04 g, 9.8 mmol). The reaction mixture was stirred at roomtemperature for 2 h, then was diluted with water (60 mL), and extractedwith EtOAc (15 mL×4). The combined organic layers were washed with brine(13 mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo.The crude residue was purified by column chromatography on silica gel(DCM:MeOH=60:1˜45:1) to give a mixture of 1783-C and 1783-C1 (380 mg,92%) as a yellow solid. MS 417.0 [M+H]⁺.

Synthesis of 1783-D and 1783-D1.

A mixture of 1783-C and 1783-C1 (380 mg, 0.91 mmol),2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole (278mg, 1.09 mmol) and K₂CO₃ (264 mg, 1.91 mmol) in dioxane/H₂O (14 mL/2 mL)was treated with Pd(PPh₃)₄ (81 mg, 0.07 mmol) under a N₂ atmosphere. Thereaction mixture was stirred at 40° C. overnight, and was thenconcentrated in vacuo. The crude residue was dissolved with DCM (50 mL),washed with brine (15 mL×3), the organic layer was dried over anhydrousNa₂SO₄ and then concentrated in vacuo. The crude residue was purified bycolumn chromatography on silica gel (DCM:MeOH=50:1˜30:1) to give amixture 1783-D and 1783-D1 (240 mg, 61%) as a brown solid. MS 436.2[M+H]⁺.

Synthesis of Compound 5.

A mixture of 1783-D and 1783-D1 (240 mg, 0.55 mmol) and Pd/C (200 mg) inMeOH/DCM (30 mL/10 mL) was stirred under a H₂ atmosphere (1 atm) at roomtemperature for 1 h. The Pd/C was then removed by filtration throughCelite, the filtrate was concentrated, and the resulting crude residuewas purified via HPLC to separate the regioisomers using chiralchromatography (Column: Chiralcel OJ-3; Solvent: MeOH; Flow rate: 2mL/min; RT₁₇₈₃=2.146 min, RT_(1641A)=1.363 min) to give Compound 5 (32mg, 21%) as a white solid. MS 406.1 [M+H]⁺.

Example 6

Synthesis of SM-C.

A mixture of 6-chloro-3-nitropyridin-2-amine (4.58 g, 26.4 mmol),2,4-difluorophenylboronic acid (5.00 g, 31.7 mmol) and Cs₂CO₃ (25.73 g,79.2 mmol) in dioxane/H₂O (100 mL/10 mL) was treated with Pd(PPh₃)₄(1.10 g, 0.95 mmol) under a N₂ atmosphere. The mixture was stirred at100° C. for 2 h and then concentrated in vacuo. The residue wasdissolved with EtOAc (200 mL) and the solution was washed with brine(100 mL×3). The organic layer was dried over anhydrous Na₂SO₄ and thenconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (PE:EtOAc=7:1˜5:1) to give SM-C (4.0 g, 61%) as a yellowsolid. MS 252.1 [M+H]⁺.

Synthesis of SM-D.

A solution of SM-C (4.0 g, 15.94 mmol) in pyridine (60 mL) was cooled to0° C., and then phenyl carbonochloridate (7.50 g, 47.81 mmol) was addeddropwise. After the addition was completed, the mixture was heated to50° C., and was stirred at 50° C. for 4 h. The mixture was then cooledto room temperature and was concentrated in vacuo. The crude residue waspurified by column chromatography on silica gel (PE:DCM=3:2˜1:1) to giveSM-D (7.1 g, 91%) as a yellow solid. MS 492.1 [M+H]⁺.

Synthesis of 1713-A.

A solution of tert-butyl 4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate (205 mg, 0.98 mmol) in HCl/dioxane (4N, 8 mL)was stirred at room temperature for 1 h. The solvent was removed invacuo to give 1713-A as a crude product which was taken on to the nextstep without further purification. MS 110.1 [M+H]⁺.

Synthesis of 1713-B.

A mixture of 1713-A (crude product from last step) and SM-D (400 mg,0.82 mmol) in DMSO (8 mL) was treated with Na₂CO₃ (691 mg, 6.52 mmol),and the resulting reaction mixture was stirred at room temperature for 2h. The mixture was then diluted with water (20 mL), and extracted withEtOAc (20 mL×3). The combined organic layers were washed with brine (20mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo. Theresidue was purified by column chromatography on silica gel(DCM:MeOH=100:1˜50:1) to give 1713-B (270 mg, 86%) as a yellow solid. MS387.1 [M+H]⁺.

Synthesis of 1713-C and 1713-C1.

A solution of 1713-B (215 mg, 0.56 mmol) in DMF (3 mL) was cooled to 0°C. and then was treated with NaH (60% in mineral oil) (67 mg, 1.68mmol). The resulting reaction mixture was allowed to warm to roomtemperature, and was stirred at room temperature for 30 min. After 30min, 3-bromooxetane (92 mg, 0.67 mmol) was added, and the reactionmixture was stirred at 50° C. overnight. The mixture was then cooled toroom temperature and was quenched with water (20 mL), then extractedwith EtOAc (20 mL×3). The combined organic layers were washed with brine(10 mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo.The residue was purified by column chromatography on silica gel(DCM:MeOH=100:1˜30:1) to give a mixture 1713-C and 1713-C1 (65 mg, 26%)isolated as a yellow solid. MS 443.1 [M+H]⁺.

Synthesis of Compound 6 and T-1713A.

A mixture of 1713-C and 1713-C1 (65 mg, 0.15 mmol) and Pd/C (65 mg) inMeOH (5 mL) was stirred under a H₂ atmosphere (1 atm) at roomtemperature for 1 h. The Pd/C was then removed by filtration throughCelite, the filtrate was concentrated, and the crude residue waspurified by HPLC, using chiral chromatography (Column: Chiralcel OD-3;Solvent: MeOH; Flow rate: 2 mL/min; RT₁₇₁₃=2.45 min, RT_(1713A)=3.99min) to give Compound 6 (10 mg, 16%) as a white solid (MS 413.2 [M+H]⁺)and T-1713A (5 mg, 8%) as a white solid. MS 413.1 [M+H]⁺.

Example 7

Synthesis of 1722-A.

To a solution of (3R,4S)-tert-butyl 3,4-diaminopyrrolidine-1-carboxylate(3.3 g, 16.4 mmol) in anhydrous ethanol (80 mL) was added ethylacetimidate hydrochloride (2.65 g, 21.3 mmol), and the reaction mixturewas stirred at 80° C. for 2.5 h. The mixture was evaporated in vacuo andthe residue was purified by column chromatography on silica gel(DCM:MeOH=50:1˜20:1) to give the 1722-A (3.0 g, 81%) as a brown oil. MS226.4 [M+H]⁺.

Synthesis of 1722-B.

To a stirred solution of oxalyl chloride (2.69 g, 21.3 mmol) in DCM (48mL) at −78° C. was added a solution of DMSO (3.16 g, 42.6 mmol) in DCM(24 mL) dropwise. The resulting reaction mixture was stirred for 10 min,and then a solution of 1722-A (3 g, 13.3 mmol) in DCM (24 mL) was addeddropwise over 10 min, followed by triethylamine (6.73 g, 66.7 mmol),which was added dropwise over 10 min. The reaction mixture was stirredwith gradual warming for 1 h. The reaction was then quenched with water(100 mL), and the organic phase was washed with brine (30 mL×3), driedover Na₂SO₄ and concertrated in vacuo. The crude residue was purified byflash chromatography on silica gel (DCM:MeOH=50:1˜20:1) to give 1722-B(1.5 g, 51%) as a white solid. MS 224.4 [M+H]⁺.

Synthesis of 1722-C.

A solution of 1722-B (300 mg, 1.35 mmol) in DMF (6 mL) was cooled to 0°C., and then treated with NaH (60% in mineral oil) (81 mg, 2.03 mmol).The reaction was allowed to warm to room temperature, and was stirred atroom temperature for 30 min. After 30 min, MeI (383 mg, 2.70 mmol) wasadded to the reaction mixture, and stirring at room temperature wascontinued for 2 h. The reaction mixture was then quenched with water (50mL), extracted with EtOAc (30 mL×3), and the combined organic layerswere washed with brine (10 mL×3), dried over anhydrous Na₂SO₄ and thenconcentrated in vacuo to give 1722-C (350 mg) as a crude product whichwas taken on without further purification. MS 238.2 [M+H]⁺.

Synthesis of 1722-D.

To a solution of 1722-C (350 mg, crude product from last step) in DCM (6mL) cooled to 0° C. was added TFA (3 mL) dropwise. The resultingreaction mixture was stirred at room temperature for 1 h, and thesolvent was removed in vacuo to give 1722-D (360 mg) as a crude productwhich was taken on to the next step without further purification. MS138.4 [M+H]⁺.

Synthesis of 1722-E.

A mixture of 1722-D (150 mg, crude product from last step) and SM-B (249mg, 0.53 mmol) in DMSO (5 mL) was treated with Na₂CO₃ (561 mg, 5.3mmol), and the reaction mixture was stirred at room temperature for 2 h.The mixture was then diluted with water (30 mL), extracted with EtOAc(20 mL×3), and the combined organic layers were washed with brine (10mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo. Theresidue was purified by Prep-TLC (EA:PE=5:1) to give 1722-E (30 mg, 14%)as a light yellow solid. MS 397.2 [M+H]⁺.

Synthesis of Compound 7.

A mixture of 1722-E (30 mg, 0.076 mmol) and Pd/C (10 mg) in MeOH (4 mL)was stirred under a H₂ atmosphere (1 atm) at room temperature for 1 h.The Pd/C was then removed by filtration through Celite, the filtrate wasconcentrated, and the residue was purified by Prep-TLC (EA:MeOH=10:1) togive Compound 7 (5 mg, 18%) as a light yellow solid. MS 3672 [M+H]⁺.

Example 8

Synthesis of 1752-A.

A mixture of 1622-B (300 mg, 0.658 mmol) and6-chloro-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine hydrochloride (151 mg,0.789 mmol) in DMSO (10 mL) was treated with Na₂CO₃ (558 mg, 5.263 mmol)and the reaction mixture was stirred at 25° C. for 2 h. The mixture wasthen diluted with water (20 mL), and extracted with EtOAc (20 mL×3). Thecombined organic layers were washed with brine (20 mL×3), dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The residue waspurified by column chromatography on silica gel (DCM:MeOH=100:1˜50:1) togive 1752-A (200 mg, 77%) as a yellow solid. MS 397.1 [M+H]⁺.

Synthesis of 1752-B.

A mixture of 1752-A (200 mg, 0.50 mmol), potassiumtrifluoro(vinyl)borate (135 mg, 1.01 mmol), X-phos (12 mg, 0.025 mmol)and Cs₂CO₃ (493 mg, 1.51 mmol) in 1,4-dioxane/H₂O (17 mL/3 mL) wastreated with Pd(OAc)₂ (3 mg, 0.01 mmol) under a N₂ atmosphere. Themixture was stirred at 80° C. for 4 h, at which point LCMS indicated thereaction had gone to completion. The reaction mixture was then extractedwith DCM (15 mL×3), and the combined organic layers were dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The residue waspurified by column chromatography on silica gel (DCM:MeOH=50:1˜12:1) togive 1752-B (100 mg, 51%) as a yellow solid. MS 388.1 [M+H]⁺.

Synthesis of Compound 8.

A mixture of 1752-B (100 mg, 0.257 mmol) and Pd/C (100 mg) in MeOH (10mL) was stirred under a H₂ atmosphere (1 atm) at 25° C. for 1 h. ThePd/C was then removed by filtration through Celite, the filtrate wasconcentrated, and the residue was purified by Prep-TLC (DCM:MeOH=10:1)to give Compound 8 (40 mg, 43%) as a gray solid. MS 361.2 [M+H]⁺.

Example 9

Synthesis of SM-C.

A mixture of 6-chloro-3-nitropyridin-2-amine (4.58 g, 26.4 mmol),2,4-difluorophenylboronic acid (5.00 g, 31.7 mmol) and Cs₂CO₃ (25.73 g,79.2 mmol) in dioxane/H₂O (100 mL/10 mL) was treated with Pd(PPh₃)₄(1.10 g, 0.95 mmol) under a N₂ atmosphere. The mixture was stirred at100° C. for 2 h and then concentrated in vacuo. The residue wasdissolved with EtOAc (200 mL) and the resulting solution was washed withbrine (100 mL×3). The organic layer was dried over anhydrous Na₂SO₄ andthen concentrated in vacuo. The residue was purified by columnchromatography on silica gel (PE:EtOAc=7:1˜5:1) to give SM-C (4.0 g,61%) as a yellow solid. MS 252.1 [M+H]⁺.

Synthesis of SM-D.

A stirred solution of SM-C (4.0 g, 15.94 mmol) in pyridine (60 mL) wasadded phenyl carbonochloridate (7.50 g, 47.81 mmol) dropwise at 0° C.After the addition was completed, the mixture was stirred at 50° C. for4 h. The mixture was concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (PE:DCM=3:2˜1:1) to give SM-D (7.1g, 91%) as a yellow solid. MS 492.1 [M+H]⁺.

Synthesis of 1667-1.

A solution of tert-butyl 4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate (205 mg, 0.98 mmol) in HCl/EA (4N, 8 mL) wasstirred at room temperature for 1 h. The solvent was removed in vacuo togive 1667-1 as a crude product which was taken on to the next stepwithout purification. MS 110.1 [M+H]⁺.

Synthesis of 1667-2.

A mixture of 1667-1 (crude product from last step) and SM-D (400 mg,0.82 mmol) in DMSO (8 mL) was treated with Na₂CO₃ (691 mg, 6.52 mmol),and the resulting reaction mixture was stirred at room temperature for 2h. The mixture was diluted with water (20 mL), extracted with EtOAc (20mL×3). The combined organic layers were washed with brine (20 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel(DCM:MeOH=100:1˜50:1) to give 1667-2 (270 mg, 86%) as a yellow solid. MS387.1 [M+H]⁺.

Synthesis of 1667-3 and 1667-3A.

A solution of 1667-2 (270 mg, 0.70 mmol) in DMF (5 mL) was cooled to 0°C. and then treated with NaH (60% in mineral oil) (84 mg, 2.10 mmol).The resulting reaction mixture was allowed to warm to room temperature,and then was stirred at room temperature for 30 min. After 30 min,1-bromo-2-methoxyethane (194 mg, 1.40 mmol) was added to the reactionmixture, and stirring was continued at room temperature for 2 h. Themixture was then quenched with water (15 mL), and extracted with EtOAc(20 mL×3). The combined organic layers were washed with brine (10 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel(DCM:MeOH=100:1˜30:1) to give a mixture of 1667-3 and 1667-3A (300 mg,97%) as a yellow solid. MS 445.0 [M+H]⁺.

Synthesis of Compound 9 and T-1667A.

A mixture of 1667-3 and 1667-3A (300 mg, 0.68 mmol) and Pd/C (300 mg) inMeOH (15 mL) was stirred at room temperature for 1 h under a H₂atmosphere (1 atm). The Pd/C was then removed by filtration throughCelite, the filtrate was concentrated, and the residue was purified bycolumn chromatography on silica gel (DCM:MeOH=100:1˜30:1) to giveCompound 8 and T-1667A. The mixture was then further purified by HPLCusing chiral chromatography (Column: Chiralcel OJ-3; Solvent:MeOH/MeCN=1/1; Flow rate: 2 mL/min; RT₁₆₆₇=1.74 min, RT_(1667A)=0.93min) to give Compound 9 (80 mg, 28%) as a white solid (MS 415.1 [M+H]′)and T-1667A (40 mg, 14%) as a white solid. MS 415.1 [M+H]⁺.

Example 10—Intentionally Omitted Example 11

Synthesis of 1621-A and 1621-A1.

A mixture of tert-butyl4,6-dihydropyrrolo[3,4-c]pyrazole-5(2H)-carboxylate (350 mg, 1.67 mmol)and CH₃I (474 mg, 3.34 mmol) in dry DMF (5 mL) was cooled to 0° C., andthen was treated with NaH (100 mg, 2.5 mmol, 60% w/w). The reactionmixture was allowed to warm to room temperature, and then was stirred atroom temperature for 3 h, whereupon it was poured into ice water (30 mL)to quench. The mixture was extracted with EtOAc (3×12 mL), and thecombined organic layers were combined and washed with brine (20 mL),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel (PE:EtOAc=4:1˜3:2)to give a mixture of 1621-A and 1621-A1 (355 mg, 95.1%) as a colorlessoil.

Synthesis of 1621-B and 1621-B1.

A mixture of 1621-A and 1621-A1 (355 mg, 1.59 mmol) was dissolved in HClin EtOAc (4M, 12 mL), and the reaction mixture was stirred at roomtemperature for 1 h. When LCMS showed the reaction was finished, thesolvent was removed in vacuo to give 1621-B and 1621-B1 (307 mg, 98.4%)as white solid which was taken on to the next step without furtherpurification.

Synthesis of 1621-C and 1621-C1.

A mixture of 1621-B and 1621-B1 (270 mg, 1.38 mmol) and 1622-B (360 mg,0.79 mmol) in DMSO (6 mL) was stirred at room temperature for 10 min,then Na₂CO₃ (670 mg, 6.32 mmol) was added, and the reaction mixture wasstirred at room temperature for 2 h. After the reaction had gone tocompletion as indicated by LCMS, the mixture was diluted with water (40mL) and extracted with EtOAc (3×12 mL). The combined organic layers werewashed with brine (3×10 mL), dried over anhydrous Na₂SO₄ and thenconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (DCM:MeOH=100:1˜60:1) to give a mixture of 1621-C and1621-C1 (230 mg, 79.8%) as a yellow solid.

Synthesis of Compound 11 and 1621A.

A mixture of 1621-C and 1621-C1 (230 mg, 0.63 mmol) and Pd/C (230 mg) inMeOH (20 mL) was stirred at room temperature for 50 min under a H₂atmosphere (1 atm). The Pd/C was then removed by filtration throughCelite, the filtrate was concentrated, and the residue was purified bychiral HPLC (Column: Chiralcel OD-3; Solvent: MeOH; Flow rate: 2 mL/min;RT₁₆₂₁=2.25 min, RT_(1621A)=3.07 min) to give Compound 11 (80 mg, 56.8%)as yellow solid and 1621A (40 mg, 56.8%) as yellow solid.

Example 12

Synthesis of 1622-A.

A mixture of 6-chloro-3-nitropyridin-2-amine (9.00 g, 51.87 mmol),pyridin-3-ylboronic acid (7.66 g, 62.25 mmol) and Cs₂CO₃ (50.70 g, 115.6mmol) in dioxane/H₂O (200 mL/20 mL) was treated with Pd(PPh₃)₄ (3.00 g,2.59 mmol) under a N₂ atmosphere. The mixture was stirred at 95° C. for3 h and then concentrated in vacuo. The residue was dissolved in EtOAc(200 mL) and then water (200 mL) was added, and the layers wereseparated. The aqueous layer was extracted with EtOAc (100 mL×3), andthe combined organic layers were washed with brine (100 mL×3) dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The residue waspurified by column chromatography on silica gel (PE:EtOAc=5:1˜3:1) togive 1622-A (7.0 g, 64%) as a yellow solid

Synthesis of 1622-B.

To a solution of 1622-A (2.00 g, 9.26 mmol) in pyridine (20.0 mL) wasadded phenyl carbonochloridate (4.33 g, 27.78 mmol) dropwise. After theaddition was completed, the mixture was heated to 50° C. and stirred for4 h. The mixture was then cooled to room temperature and concentrated invacuo. The residue was purified by column chromatography on silica gel(DCM:EtOAc=8:1˜2:1) to give 1622-B (3.80 g, 90%) as a yellow solid.

Synthesis of 1622-C.

A solution of of 6-chloro-2,3-dihydro-1H-pyrrolo[3,4-c]pyridinehydrochloride (5.00 g, 26.2 mmol) in DCM (200 mL) was cooled to 0° C.and treated with DIPEA (6.75 g, 52.4 mmol), added slowly, and thereaction mixture was stirred at 0° C. for 0.5 h. Then (Boc)₂O was addedslowly, and after the addition was complete the reaction was allowed towarm to room temperature and stirred for 1 h. After 1 h, the reactionhad gone to completion as indicated by when LCMS, at which point thereaction mixture was diluted with water, and extracted with DCM, driedover Na₂SO₄, concentrated under reduced pressure and purified by flashcolumn chromatography to obtain 1622-C (5.5 g, 83%) as white solid.

Synthesis of 1622-D.

A mixture of 1622-C (320 mg, 1.38 mmol), trimethylboroxine (522 mg, 4.14mmol) and K₂CO₃ (952 mg, 6.9 mmol) in dioxane (15 mL) was treated withPd(dppf)₂Cl₂ (57 mg, 0.07 mmol) under a N₂ atmosphere. The mixture wasstirred at 95° C. for 24 h and then concentrated in vacuo. The residuewas dissolved with EtOAc (20 mL) and the solution was washed with brine(20 mL×3). The organic layer was dried over anhydrous Na₂SO₄ and thenconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (PE:EtOAc=5:1˜2:1) to give 1622-D (160 mg, 50%) as a whitesolid.

Synthesis of 1622-E.

A solution of 1622-D (160 mg, 0.68 mmol) in DCM (5 mL) was treated withTFA (2 mL) and stirred at room temperature for 1 h, at which point LCMSindicated that the reaction was finished. The solvent was then removedin vacuo to give 1622-E as a crude product which was used directly inthe next step.

Synthesis of 1622-F.

A mixture of 1622-B (190 mg, 0.42 mmol) and 1622-E (crude product fromlast step) in DMSO (5 mL) was stirred at room temperature for 10 min,then Na₂CO₃ (222 mg, 2.1 mmol) was added and the reaction mixture wasstirred at room temperature for 2 h. After the reaction was completed asindicated by LCMS, the mixture was diluted with water (30 mL) andextracted with EtOAc (10 mL×3). The combined organic layers were washedwith brine (10 mL×3), dried over anhydrous Na₂SO₄ and then concentratedin vacuo. The residue was purified by column chromatography on silicagel (DCM:MeOH=100:1˜50:1) 1622-F (100 mg, 63%) as a yellow solid

Synthesis of Compound 12.

A mixture of 1622-F (100 mg, 0.32 mmol) and Pd/C (100 mg) in MeOH (10mL) was stirred at room temperature for 30 min under a H₂ atmosphere (1atm). The Pd/C was then removed by filtration through Celite, thefiltrate was concentrated and the residue was purified by Prep-TLC(DCM:MeOH=10:1) to give Compound 12 (52 mg, 56%) as a white solid.

Example 13—Intentionally Omitted Example 14

Synthesis of 1542-A.

A solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (30.0 g, 162.0mmol) and DMF-DMA (58.0 g, 486.5 mmol) in THF (300 mL) was stirred at70° C. for 12 h. The reaction mixture was then concentrated to give1542-A as a crude product which was used directly in the next step. MS241.2 [M+H]⁺.

Synthesis of 1542-B.

A mixture of 1542-A (162.0 mmol, crude product from last step),acetimidamide hydrochloride (61.0 g, 648.0 mmol) and Et₃N (65.0 g, 648.0mmol) in EtOH (450 mL) was stirred at 80° C. for 16 h. The mixture wasthen cooled to room temperature and diluted with water (500 mL), thenextracted with EtOAc (400 mL×3). The combined organic layers were washedwith brine (100 mL×3), dried over anhydrous Na₂SO₄ and then concentratedin vacuo. The residue was purified by column chromatography on silicagel (PE:EtOAc=10:1˜1:1) to give 1542-B (16.0 g, 42%) as a gray solid. MS236.1 [M+H]⁺.

Synthesis of 1542-C.

To a solution of 1542-B (16.0 g, 68.0 mmol) in DCM (100 mL) cooled to 0°C. was added TFA (100 mL) dropwise. The reaction mixture was allowed towarm to room temperature, and then stirred at room temperature for 1 h.The solvent was removed in vacuo to give 1542-C as a crude product whichwas used directly in the next step. MS 136.0 [M+H]⁺.

Synthesis of 1542-D.

To a solution of SM-B (16.0 g, 33.8 mmol) and 1542-C (68.0 mmol, crudeproduct from last step) in DMSO (160 mL) was added Na₂CO₃ (35.0 mg, 338mmol). The resulting mixture was stirred at room temperature for 3 h.The mixture was then diluted with water (500 mL) and the precipitate wascollected by filtration. The filter cake was recrystallized from DCM(200 mL) to provide 1542-D (9.4 g, 70%) as a brown solid. MS 395.4[M+H]⁺.

Synthesis of Compound 14.

To a solution of 1542-D (9.4 g, 23.8 mmol) and Pd/C (1.88 g) in DCM/MeOH(200 mL/200 mL) was stirred at room temperature for 2 h under anatmosphere of H₂ (1 atm). The Pd/C was then removed by filtrationthrough Celite, the filtrate was concentrated, and the residue waspurified by column chromatography on silica gel (MeOH:EtOAc=0:1˜10:1) togive Compound 14 (3.8 g, 44%) as a gray solid. MS 365.4 [M+H]⁺.

Example 15

Synthesis of 1791-1.

A solution of prop-2-yn-1-amine (5.0 g, 90.9 mmol) and Et₃N (18.4 g,181.8 mmol) in DCM (100 mL) was cooled to 0° C., and then treated with(Boc)₂O (23.8 g, 109.1 mmol). The resulting reaction mixture was allowedto warm to room temperature, and was stirred at room temperature for 16h. The mixture was then diluted with DCM (200 mL), washed with brine(100 mL×3), and the organic layer was dried over Na₂SO₄ and thenconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (PE:EtOAc=100:1˜10:1) to give 1791-1 (10 g, 71%) as acolorless oil. MS 178.3 [M+23]⁺, 100.3 [M−56]⁺.

Synthesis of 1791-2.

A solution of 1791-1 (10 g, 64.5 mmol) in DMF (200 mL) was cooled to 0°C. and treated with NaH (60% in mineral oil) (2.84 g, 71 mmol), addedslowly. The resulting reaction mixture was allowed to warm to roomtemperature, and was stirred at room temperature for 1 h. Then3-bromoprop-1-yne (9.2 g, 77.4 mmol) was added to the reaction mixture,and stirring was continued at room temperature for 2 h. The mixture wasthen quenched with water (500 mL), and extracted with t-BuOMe (250mL×3). The combined organic layers were washed with brine (200 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel(PE:EtOAc=100:1˜10:1) to give 1791-2 (12 g, 96%) as a yellow oil. MS138.1 [M−56]⁺.

Synthesis of 1791-3.

To a solution of ethyl carbonocyanidate (4.10 g, 41.4 mmol) and[Cp*RuCl(cod)] (394 mg, 1.0 mmol) in DCE (40 mL) was added a solution of1791-2 (4.0 g, 20.7 mmol) in DCE (80 mL) dropwise over 30 min under a N₂atmosphere. The resulting mixture was stirred at 40° C. for 16 h. Thesolvent was then removed in vacuo, and the residue was purified bycolumn chromatography on silica gel (PE:EtOAc=10:1˜2:1) to give 1791-3(2.1 g, 35%) as a tan solid. MS 293.3 [M+H]⁺.

Synthesis of 1791-4.

To a solution of 1791-3 (2.0 g, 6.8 mmol) in anhydrous ethanol was addedNaBH₄ (1.56 g, 41.1 mmol) at room temperature. The reaction mixture wasstirred at room temperature for 16 h, whereupon the mixture was quenchedwith water (50 mL), and extracted with DCM (30 mL×3). The combinedorganic layers were washed with brine (30 mL×3), dried over anhydrousNa₂SO₄ and then concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (PE:EtOAc=50:1˜5:1) to give 1791-4(1.2 g, 70%) as a white solid. MS 251.1 [M+H]⁺.

Synthesis of 1791-A.

A mixture of 1791-4 (500 mg, 2.0 mmol) and Dess-Martin Periodinane (1.7g, 4.0 mmol) in DCM (20 mL) was stirred at 30° C. for 24 h. Theprecipitate was then removed by filtration through Celite, and thefiltrate was concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel (DCM:MeOH=100:1˜50:1) togive 1791-A (450 mg, 91%) as a white solid. MS 149.3 [M−100]⁺.

Synthesis of 1791-B.

A mixture of sodium 2-chloro-2,2-difluoroacetate (228 mg, 1.8 mmol) andPPh₃ (472 mg, 1.8 mmol) in DMF (10 mL) was stirred at room temperaturefor 2 h. Then 1791-A (300 mg, 1.2 mmol) was added, and the resultingmixture was heated to 100° C. and stirred at 100° C. for 1 h. Themixture was then cooled to room temperature and diluted with water (30mL), then extracted with EtOAc (20 mL×3). The combined organic layerswere washed with brine (20 mL×3), dried over anhydrous Na₂SO₄ and thenconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (DCM:MeOH=100:1˜30:1) to give 1791-B (220 mg, 60%) as awhite solid. MS 283.3 [M+H]⁺.

Synthesis of 1791-C.

A mixture of 1791-B (200 mg, 0.71 mmol) and Pd/C (120 mg) in EtOAc (15mL) was stirred under a H₂ atmosphere (1 atm) at room temperature for 40min. The Pd/C was then removed by filtration through Celite, and thefiltrate was concentrated to give 1791-C (170 mg, 84.4%) as a brownsolid. MS 285.2 [M+H]⁺.

Synthesis of 1791-D.

To a solution of 1791-C (170 mg, 0.60 mmol) in DCM (6 mL) was added TFA(1.5 mL). Then the solution was stirred at room temperature for 0.5 h.The solvent was removed in vacuo to give 1791-D as a crude product whichwas taken on to the next step without purification. MS 185.2 [M+H]⁺.

Synthesis of 1791-E.

A solution of 1791-D (0.60 mmol, crude product from last step) and1622-B (143 mg, 0.31 mmol) in DMSO (5 mL) was treated with Na₂CO₃ (394mg, 3.72 mmol), and the mixture was stirred at room temperature for 2 h.The reaction mixture was then diluted with water (20 mL), extracted withEtOAc (10 mL×4), the combined organic layers were washed with brine (10mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo. Theresidue was purified by column chromatography on silica gel(DCM:MeOH=50:1˜30:1) to give 1791-E (80 mg, 60%) as a yellow solid. MS427.1 [M+H]⁺.

Synthesis of Compound 15.

A mixture of 1791-E (80 mg, 0.19 mmol) and Pd/C (100 mg) in MeOH/EtOAc(8 mL/8 mL) was stirred under a H₂ atmosphere (1 atm) at roomtemperature for 40 min. The Pd/C was then removed by filtration throughCelite, the filtrate was concentrated and the residue was purified byPrep-HPLC to give Compound 15 (33 mg, 44%) as white solid. MS 397.2[M+H]⁺.

Example 16

Synthesis of 1792-A.

To a solution of 1791-A (125 mg, 0.5 mmol) in DCM (5 mL) was added DAST(201 mg, 1.25 mmol) dropwise at −78° C. The reaction mixture was thenwarmed to room temperature and stirred for 3 h. The mixture was thendiluted with DCM (15 mL), washed with brine (10 mL×3), dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The residue waspurified by column chromatography on silica gel (DCM:MeOH=100:1˜30:1) togive 1792-A (110 mg, 81%) as a white solid. MS 271.2 [M+H]⁺.

Synthesis of 1792-B.

A solution of 1792-A (110 mg, 0.41 mmol) in DCM (4 mL) was cooled to 0°C. and then TFA (2 mL) was added dropwise. The reaction mixture wasallowed to warm to RT and stirred at room temperature 1 h. The solventwas removed in vacuo to give 1792-B as a crude product which was takenon to the next step without purification. MS 171.4 [M+H]⁺.

Synthesis of 1792-C.

To a mixture of 1792-B (0.41 mmol, crude product from last step) and1622-B (186 mg, 0.41 mmol) in DMSO (5 mL) was added Na₂CO₃ (217 mg, 2.05mmol), and the reaction mixture was stirred at 25° C. for 2 h. Themixture was then diluted with water (20 mL), extracted with EtOAc (10mL×3), and the combined organic layers were washed with brine (10 mL×3),dried over anhydrous Na₂SO₄ and then concentrated in vacuo. The residuewas purified by column chromatography on silica gel(DCM:MeOH=100:1˜30:1) to give 1792-C (110 mg, 67%) as a yellow solid. MS413.2 [M+H]⁺.

Synthesis of Compound 16.

A mixture of 1792-C (110 mg, 0.27 mmol) and Pd/C (110 mg) in MeOH/EtOAc(5 mL/5 mL) was stirred under a H₂ atmosphere (1 atm) at roomtemperature for 1 h. The Pd/C was then removed by filtration throughCelite, the filtrate was concentrated and the residue was purified byPrep-TLC (DCM:MeOH=15:1) to give Compound 16 (50 mg, 48%) as a yellowsolid. MS 383.2 [M+H]⁺.

Example 17

Synthesis of 1687-A.

A solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (4.0 g, 21.6mmol) and DMF-DMA (7.6 g, 64.8 mmol in THF (40 mL) was stirred at 70° C.for 16 h. The solution was concentrated in vacuo to give 1687-A as acrude product which was used directly in the next step. MS 241.3 [M+H]⁺.

Synthesis of 1687-B.

To a solution of 1687-A (8.2 mmol, crude product from last step) in EtOH(10 mL) was added Et₃N (4.1 g, 41.0 mmol) and propionimidamidehydrochloride (3.55 g, 32.8 mmol). The resulting solution was stirred at80° C. for 24 h. After the mixture was cooled to room temperature, themixture was diluted with water (50 mL) and extracted with DCM (30 mL×3).The combined organic layers were washed with brine (30 mL×3), dried overanhydrous Na₂SO₄ and then concentrated in vacuo. The residue waspurified by column chromatography on silica gel (PE:DCM=10:1˜1:2) togive 1687-B (1.0 g, 49%) as a brown solid. MS 250.3 [M+H]⁺.

Synthesis of 1687-C.

To a solution of 1687-B (1.0 g, 4.02 mmol) in DCM (10 mL) was added TFA(5 mL) in dropwise fashion. The reaction mixture was stirred at roomtemperature for 1 h, then was concentrated in vacuo to give 1687-C as acrude product which was used directly in the next step. MS 150.3 [M+H]⁺.

Synthesis of 1687-D.

A mixture of SM-B (300 mg, 0.63 mmol) and 1687-C (0.82 mmol, crudeproduct from last step) in DMSO (8 mL) was treated with Na₂CO₃ (537 mg,5.07 mmol), and the reaction mixture was stirred at room temperature for2 h. The mixture was then diluted with water (20 mL), and extracted withEtOAc (20 mL×3). The combined organic layers were washed with brine (20mL×3), dried over anhydrous Na₂SO₄ and then concentrated in vacuo. Theresidue was purified by column chromatography on silica gel(DCM:MeOH=100:1˜50:1) to give 1687-D (150 mg, 58%) as a yellow solid. MS409.4 [M+H]⁺.

Synthesis of Compound 17.

A mixture of 1687-D (100 mg, 0.25 mmol) and Pd/C (100 mg) in MeOH (10mL) was stirred under a H₂ atmosphere (1 atm) at room temperature for 1h. The Pd/C was then removed by filtration through Celite, the filtratewas concentrated and the residue was purified by Prep-TLC(DCM:MeOH=10:1) to give Compound 17 (25 mg, 27%) as a yellow solid (MS379.4 [M+H^(]+)).

TABLE 1 Spectrometric Data for Compounds MS MS ¹H NMR Data (400 MHz, No.Structure Calc found DMSO-d₆) 1

394 395 δ 8.49 (s, 1H), 7.98 (dd, J = 8.8, 5.6 Hz, 2H), 7.75 (s, 1H),7.57 (d, J = 8.2 Hz, 1H), 7.23 (t, J = 8.8 Hz, 2H), 7.18 (d, J = 8.0 Hz,1H), 5.62-5.55 (m, 1H), 5.17 (s, 2H), 4.93-4.90 (m, 4H), 4.56 (d, J =21.6 Hz, 4H). 2

370 371 δ 8.70 (s, 1H), 8.58 (s, 1H), 7.51(d, J = 8.0 Hz, 1H), 7.17 (t,J = 4.0 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.68-6.66 (m, 1H), 5.20 (s,2H), 4.76 (d, J = 13.2 Hz, 4H), 2.64 (s, 3H). 3

366 367 δ 8.73 (s, 1H), 8.59 (s, 1H), 7.58- 7.46 (m, 2H), 7.41 (dd, J =5.0, 1.0 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 7.06 (dd, J = 5.0, 3.7 Hz,1H), 5.18 (s, 2H), 4.77 (d, J = 8.2 Hz, 4H), 2.92 (q, J = 7.6 Hz, 2H),1.29 (t, J = 7.6 Hz, 3H). 4

355 356 δ 8.43 (s, 1H), 7.99 (s, 1H), 7.55- 7.51 (m, 2H), 7.13 (d, J =8.2 Hz, 1H), 5.24 (s, 2H), 4.49 (s, 4H), 3.85 (s, 3H), 2.62 (s, 3H). 5

405 406 δ 8.47 (s, 1H), 7.99 (s, 1H), 7.65 (s, 1H), 7.52 (d, J = 8.0 Hz,1H), 7.12 (d, J = 8.0 Hz, 1H), 6.36 (tt, J = 55.2, 4.0 Hz, 1H), 5.24 (s,2H), 4.67-4.59 (m, 6H), 2.62 (s, 3H). 6

412 413 δ 8.51 (s, 1H), 7.94 (dd, J = 16.0, 9.0 Hz, 1H), 7.74 (s, 1H),7.41 (dd, J = 8.0, 1.9 Hz, 1H), 7.32-7.27 (m, 1H), 7.19-7.14 (m, 2H),5.72-5.55 (m, 1H), 5.26 (s, 2H), 4.94-4.88 (m, 4H), 4.55 (d, J = 21.2Hz, 4H). 7

366 367 δ 8.40 (s, 1H), 7.98 (dd, J = 8.4, 5.6 Hz, 2H), 7.57 (d, J = 8.0Hz, 1H), 7.22 (t, J = 8.2 Hz, 2H), 7.17 (d, J = 8.4 Hz, 1H), 5.14 (s,2H), 4.53-4.42 (m, 4H), 3.53 (s, 3H), 2.30 (s, 3H). 8

360 361 δ 9.14 (d, J = 1.6 Hz, 1H), 8.59 (s, 1H), 8.49-8.47 (m, 2H),8.27 (dt, J = 8.4, 2.0 Hz, 1H), 7.69-7.67 (d, J = 8.4 Hz, 1H), 7.44-7.41(m, 1H), 7.30 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 5.31 (s, 2H), 4.80 (s,4H), 2.77 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). 9

414 415 δ 8.48 (s, 1H), 7.98-7.91 (m, 1H), 7.57 (s, 1H), 7.41 (dd, J =8.0, 2.0 Hz, 1H), 7.32-7.26 (m, 1H), 7.18-7.13 (m, 2H), 5.25 (s, 2H),4.52 (s, 4H), 4.27- 4.24 (t, J = 5.4 Hz, 2H), 3.69- 3.66 (t, J = 5.2 Hz,2H), 3.24 (s, 3H). 11

355 356 δ 9.15 (s, 1H), 8.48 (s, 2H), 8.29-8.26 (m, 1H), 7.68-7.66 (d, J= 8 Hz, 1H), 7.56 (s, 1H), 7.44-7.40 (m, 1H), 7.21-7.19 (d, J = 8 Hz,1H), 5.28 (s, 2H), 4.52 (s, 4H), 3.85 (s, 3H) 12

346 347 ¹H NMR (400 MHz, CDCl₃) δ 9.11 (d, J = 1.8 Hz, 1H), 8.59- 8.55(m, 1H), 8.51 (s, 1H), 8.27- 8.08 (m, 1H), 7.47 (d, J = 8.1 Hz, 1H),7.34 (dd, J = 7.5, 4.8 Hz, 1H), 7.20 (d, J = 8.1 Hz, 1H), 7.16 (s, 1H),6.95 (s, 1H), 4.90 (m, 4H), 4.67 (s, 2H), 2.61 (s, 3H). 14

364 365 δ 8.71 (s, 1H), 8.63 (s, 1H), 7.99- 7.96 (q, J = 5.6 Hz, 2H),7.59- 7.57 (d, J = 8.0 Hz, 1H), 7.25- 7.16 (m, 3H), 5.20 (s, 2H),4.79-4.77 (d, J = 8.8 Hz, 4H), 2.64 (s, 3H). 15

396 397 δ 9.14 (m, 1H), 8.61-8.57 (m, 2H), 8.48 (m, 1H), 8.29-8.26 (m,1H), 7.69-7.67 (m, 1H), 7.44-7.40 (m, 2H), 7.20-7.18 (m, 1H), 6.44 (tt,J = 56.0, 4.8 Hz, 1H), 5.31 (s, 2H), 4.83 (s, 4H), 3.45-3.36 (m, 2H). 16

382 383 δ 9.14 (d, J = 2.0 Hz, 1H), 8.71 (s, 1H), 8.65 (s, 1H), 8.48(dd, J = 4 .4, 1.2 Hz, 1H), 8.29-8.26 (m, 1H), 7.78 (s, 1H), 7.68 (d, J= 8.4 Hz, 1H), 7.44-7.41 (m, 1H), 7.19 (d, J = 8.4 Hz, 1H), 7.00 (t, J =54.8 Hz, 1H), 5.32 (s, 2H), 4.90 (s, 4H). 17

378 379 δ 8.74 (s, 1H), 8.61 (s, 1H), 8.00-7.96 (m, 2H), 7.58 (d, J =8.2 Hz, 1H), 7.25-7.16 (m, 3H), 5.18 (s, 2H), 4.78 (d, J = 9.1 Hz, 4H),2.92 (q, J = 7.5 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H).

HDAC2 and HDAC1 Enzymatic Assay

The following describes an assay protocol for measuring thedeacetylation of a peptide substrate by the enzymes HDAC2 or HDAC1.Enzyme, substrate, and cofactors are combined in a well of a microtiterplate and incubated for 3 hours at 25° C. At the end of the incubation,the reaction is quenched by the addition of an SDS-containing buffer.Substrate and product are separated and quantified electrophoreticallyusing the microfluidic-based LabChip 3000 Drug Discovery System fromCaliper Life Sciences. The peptide substrate used in this assay isFAM-TSRHK(AC)KL-CONH2 (FAM is carboxyfluorescein). Peptide shouldbe >98% purity by Capillary Electrophoresis.

-   -   1. To a well of a 384-well plate, add 5 μL of 2× enzyme buffer.        Using Labcyte Echo 550, add 100 nl compound. Enzyme and compound        may be pre-incubated at this time if desired.    -   2. Add 5 μL of 2× substrate buffer.    -   3. Incubate plate at 25° C. for 17 hours.    -   4. Terminate reaction by adding 40 μL of 1.55× stop buffer.    -   5. Create job on a Caliper LabChip® 3000 Drug Discovery System.    -   6. Load the plate and start electrophoresis using blue laser        (480 nm) for excitation and green CCD (520 nm) for detection        (CCD2).        Reaction time=17 hours; Reaction temperature=25° C.

Final Assay Reaction Mixture

100 mM HEPES, pH 7.5 0.1% BSA 0.01% Triton X-100 25 mM KCl

1% DMSO (from compound) 1 μM FAM-TSRHK(AC)KL-CONH2 5 nM HDAC Enzyme(specific activity may vary from lot-to-lot, and enzyme concentrationmay need to be adjusted to yield˜10-20% conversion of substrate toproduct).

Substrate and product peptides present in each sample are separatedelectrophoretically using the LabChip 3000 capillary electrophoresisinstrument. As substrate and product peptides are separated, two peaksof fluorescence are observed. Change in the relative fluorescenceintensity of the substrate and product peaks is the parameter measured,reflecting enzyme activity. Capillary electrophoregramms (RDAacquisition files) are analyzed using HTS Well Analyzer software(Caliper Life Sciences). The kinase activity in each sample isdetermined as the product to sum ratio (PSR): P/(S+P), where P is thepeak height of the product peptide and S is the peak height of thesubstrate peptide. For each compound, enzyme activity is measured atvarious concentrations (12 concentrations of compound spaced by 3×dilution intervals). Negative control samples (0%-inhibition in theabsence of inhibitor) and positive control samples (100%-inhibition, inthe presence of 20 mM EDTA) are assembled in replicates of four and areused to calculate %-inhibition values for each compound at eachconcentration. Percent inhibition (P_(inh)) is determined usingfollowing equation:P_(inh)=(PSR_(0%)−PSR_(inh))/(PSR_(0%)−PSR_(100%))*100, where PSR_(inh)is the product sum ratio in the presence of inhibitor, PSR_(0%) is theaverage product sum ration in the absence of inhibitor and PSR₁₀₀% isthe average product sum ratio in 100%-inhibition control samples.

The IC50 values of inhibitors are determined by fitting the inhibitioncurves (P_(inh) versus inhibitor concentration) by 4 parameter sigmoidaldose-response model using XLfit 4 software (IBDS).

The results of this assay for certain compounds are reported in Table 2,below. In the table, “A” indicates a K_(d) value of between 0.1 μM and0.5 μM and “B” a K_(d) value of greater than 0.5 μM and less than orequal to 5.0 μM.

TABLE 2 Compound HDAC2 IC50, HDAC1 IC50, No. (uM) (uM) 1 C B 2 C B 3 C B4 C C 5 C B 6 B B 7 C C 8 B B 9 C B 11 C C 12 C C 14 C B 15 C C 16 C C17 B B

HDAC2 Enzymatic Inhibition Assay in SY5Y Cell Lysate

Cell Culture and Inhibitor Treatments

SH-SY5Y cells (Sigma) were cultured in Eagle's Modified Essential Mediumsupplemented with 10% fetal bovine serum and pen/strep. Twenty-fourhours prior to compound dosing 20 uL of cells were plated in white 384well plates at a density of 1,500 cells/well. Compounds were seriallydiluted in neat DMSO and then diluted 1:100 v/v into media without FBSand mixed. Media was removed from the plated cells and the dilutedcompounds in serum free media (1% v/v final DMSO) were added andincubated at 37.0 for five hours. Ten uL of HDAC-Glo 2 reagent with 0.1%Triton X-100 was then added, the plate was mixed and allowed to developat room temperature for 100 minutes. Plates were then read with aSpectramax LMax luminometer employing a 0.4 s integration time. Doseresponse curves were constructed with normalized data where CI-994 at100 uM was defined as 100% inhibition and DMSO alone as 0% inhibition.

The results of this assay for certain compounds are reported in Table 3,below. In the table, “A” indicates an IC₅₀ value of between 0.1 μM and 1μM; “B” indicates an a IC₅₀ value of between 1.0 μM and 1.5 μM; and “C”indicates an a IC₅₀ value of greater than 1.5 μm.

TABLE 3 HDAC2 IC50, Compound SY5Y Cell Lysate No. (uM) 1 A 2 B 3 A 4 A 5A 6 A 7 A 8 A 9 A 11 A 12 A 14 A 15 A 16 C 17 B

Erythroid and Myeloid CFU Assay

Compounds were tested to evaluate the potential effects on humanerythroid and myeloid progenitors using colony forming cell assays.Clonogenic progenitors of human erythroid (CFU-E, BFU-E),granulocyte-monocyte (CFU-GM) and multipotential (CFU-GEMM) lineageswere assessed in a semi-solid methylcellulose-based media formulationcontaining rhlL-3 (10 ng/mL), rhGM-SCF (10 ng/mL), rhSCF (50 ng/mL) andEpo (3 U/mL).

Cells

Normal human bone marrow light density cells derived from normal bonemarrow (NorCal Biologics, California), were stored in the gaseous phaseof liquid nitrogen (−152° C.) until required for the assay. On the dayof the experiment, the cells were thawed rapidly, the contents of eachvial was diluted in 10 mL of Iscove's modified Dulbecco's mediumcontaining 10% fetal bovine serum (IMDM+10% FBS) and washed bycentrifugation (approximately 1200 r.p.m. for 10 minutes, roomtemperature). The supernatant was discarded and the cell pelletsresuspended in a known volume of IMDM+10% FBS. A cell count (3% glacialacetic acid) and viability assessment (trypan blue exclusion test) wasperformed for the bone marrow sample.

Compounds

On the day of the experiment, the compounds were dissolved in DMSO to astock concentration of 10 mM. Serial dilutions were prepared from thestock concentration to achieve concentrations of 2 and 0.4 mM. Whenadded to the methylcellulose-based media at 1:1000 (v/v), the final testconcentrations of 10, 2 and 0.4 μM were achieved. Additionally, 5-FU wasevaluated at 1.0, 0.1 and 0.01 μg/mL.

Method Summary

Clonogenic progenitors of the human erythroid (CFU-E and BFU-E) andmyeloid (CFU-GM) lineages were set up in the methylcellulose-based mediaformulations described above. All compounds were added to the medium togive the final desired concentrations (10, 2 and 0.4 μM). 5-Fluorouracil(Sigma Aldrich) was used as a positive control for progenitorproliferation (inhibition of colony growth) and was introduced to thehuman bone marrow cultures at 1.0, 0.1, and 0.01 μg/mL. Solvent controlcultures (containing no compound but 0.1% DMSO) as well as standardcontrols (containing no compound and no DMSO) were also initiated.

Human myeloid and erythroid progenitor assays were initiated at 2.0×10⁴cells per culture. Following 14 days in culture, myeloid and erythroidcolonies were assessed microscopically and scored by trained personnel.The colonies were divided into the following categories based on sizeand morphology: CFU-E, BFU-E, CFU-GM and CFU-GEMM.

Statistical Analyses of CFC Numbers

The mean±one standard deviation of three replicate cultures wascalculated for progenitors of each category (CFU-E, BFU-E, etc.).Two-tailed t-tests were performed to assess if there was a difference inthe number of colonies generated between solvent control and treatedcultures. Due to the potential subjectivity of colony enumeration, a pvalue of less than 0.01 is deemed significant. To calculate theconcentration of 50% inhibition of colony growth (IC₅₀) for eachcompound, a dose response curve was generated plotting the log of thecompound concentration versus the percentage of control colony growthusing XLfit software (IDBS). The concentration of 50% inhibition ofcolony growth (IC₅₀) was calculated based on the sigmoid curve fit usingDose-Response, One-Site Model formula: y=A+[(B−A)/(1+((C/x){circumflexover ( )}D))], where A=the initial value (baseline response), B=maximumresponse, C=center (drug concentration that provokes a response halfwaybetween A and B) and D=slope of the curve at midpoint. Further, plotsand additional dose response curves were generated using GraphPad Prism7.0.

Morphological Assessment of Colonies

Photographs were taken of representative hematopoieticprogenitor-derived colonies from various lineages, illustrating coloniesin the presence of the solvent control as well as colonies in thepresence of the test compounds.

Erythroid (CFU-E and BFU-E), myeloid (CFU-GM) and multi-potential(CFU-GEMM) colony enumeration was performed by trained personnel. Thedistribution of colony types as well as general colony and cellularmorphology was analyzed. For statistical analysis colony numbers incompound treated cultures were compared to the solvent control cultures.5-FU was used as a positive control for toxicity in these assays and theinhibitory effects obtained for this compound were exactly as expected.The experiment was used to evaluate the potential effect of testcompounds on human erythroid and myeloid progenitor proliferation in amethylcellulose-based medium. The IC₅₀ values were calculated fromXLfit. Dose response curves for erythroid and myeloid toxicity generatedby XLfit. Finally, nonlinear regression curve fitting and IC₅₀s±95% CI,were calculated by Prism 7.0.-GEMM. Results are shown in Table 4.

As shown from the data, certain structural modifications were found tohave a profound effect on safety. For example, in certain instances, thereplacement of a 4-fluorophenyl group (such as in Comparator 2 andComparator 3) with more polar ring systems such as2-methyl-1,3-thiazol-5-yl (Compound 4) or pyridyl (Compounds 8, 11, and12) afforded higher % control remaining in both the erythroid andmyeloid lineage. This same safety advantage was gained by replacing the4-fluorophenyl group in pyrrolopyridine compounds (such as Comparator 3)with a 3-pyridyl group (Compound 8 and Compound 12). Installing a polarsubstituent on the pyrazole ring of the pyrrolopyrazole of Comparator 2also generated an improved safety profile along with when an oxetan-3-ylsubstituent (Compound 1) was replaced a methyl in Comparator 2. Asimilar improvement in safety profile was achieved by exchanging a4-fluorophenyl group for a 2,4-difluorophenyl group in thepyrrolopyrazole series. By, replacing the 4-fluorophenyl of Comparator 6with a 2,4-difluorophenyl group, Compound 9 also realized an improvedsafety profile, particularly in the myeloid lineage.

TABLE 4 Erythroid % Myeloid % control @ 10 control @ 10 CompoundStructure uM dose uM dose Comparator 2

15.7 30.4  4

53 96 11

94 103 Comparator 6

28 39  9

40.4 62.8 Comparator 3

25.7 66.1 12

93 104  8

98 102 Comparator 1

47.1 83.9  1

37 54 14

53 112  2

40 87  3

46.6 89.4 17

59.6 79.1 Comparator 5

22.9 58.9

Assessment of Brain and Plasma Exposure for Rodin's Compounds FollowingIntravenous (IV) and Oral (PO) Administration to Mice

Compounds were dosed in mice at either 10 mg/kg or 30 mg/kg PO, and weredosed at 1 mg/kg IV. Three animals for collection at each time point forplasma via bleeding at 0.25, 0.5, 1, 4, 12 and 24 h. Terminal bleedingfor plasma and sampling for brain at 0.25, 0.5, 1, 4, 12 and 24 h (alsothree animals per brain exposure time point group). Total of six timepoints for plasma and six time points for brain.

Sample Collection:

Plasma: The animal was restrained manually at the designated timepoints, approximately 150 μL blood/time point was collected into K₂EDTAtube via retro orbital puncture or cardiac puncture under anesthesiawith Isoflurane. The blood sample was centrifuged (2000 g, 4° C., 5 min)to generate plasma within 30 min after bleeding.

Brain: At the designated time points, a mid-line incision was made inthe animals scalp and the skin was retracted. Using small bone cuttersand rongeurs, removed the skull overlying the brain. Removed the brainusing a spatula and rinse with cold saline. Placed the brain inscrew-top tubes, and then stored the tubes under −70° C. until analysis.Results are shown in Table 5.

As shown from the data below, the addition of alkyl groups (ethyl ormethyl) to the carbon position between the pyrimidine nitrogens (e.g.,Compounds 2, 3, 14, and 17) led to increased half life (with both IV andPO dosing) and decreased clearance (IV), as well as improved brainexposure relative to unsubstituted counteparts (e.g., Comparator 1 andComparator 6).

TABLE 5 Brain Projected Cmax @ free brain @ 10 mpk 10 mpk ng/g (*scaled(uM) IV PK for (*scaled for IV PK Cl Compound Structure comparison)comparison) T_(1/2) (hr) (L/hr/kg) Comparator 1

 158* 0.111* Not available Not available Comparator 6

 100 0.103 0.152 PO = 0.557 8.33  14

 633 0.403 2.09 PO = 3.22 0.859 17

1241 0.568 2.07 PO = 2.54 1.49   3

 526 0.387 0.737 PO = 1.48 1.50   2

 760 0.438 0.643 PO = 1.25 4.06   1

 636 0.324 3.11 PO = 2.05 0.421  4

 324 0.479 1.86 PO = 2.12 0.536  5

 277 0.302 2.81 PO = 2.33 0.354  7

 102 0.050 3.06 PO = 2.95 1.71   8

 279 0.222 0.628 PO = 1.27 2.11   9

1533 1383 0.881 0.795 0.644 PO = 1.39, 1.01 1.67  11

 369 0.664 7.53 PO = 2.90 1.07  12

237 0.386 0.513 PO = 3.67 1.77  *Indicates compound dosed 30 mg/kg PO,and data has been scaled to compare to the 10 mg/kg PO data obtained forother compounds.

In Vitro Evaluation of Compounds on Human Megakaryocyte Progenitor CFCProliferation in Normal Bone Marrow

Compounds were tested to evaluate the potential effects on humanmegakaryocyte progenitor proliferation in the CFU-Mk assay. Clonogenicprogenitors of the human megakaryocyte (CFU-Mk) lineage were assessed ina semi-solid, collagen-based matrix containing rhIL-3 (10 ng/mL), rhIL-6(10 ng/mL) and rhTpo (50 ng/mL).

Cells

Normal human bone marrow light density cells (lot #0170525), derivedfrom normal bone marrow (NorCal Biologics, California), were stored at−152° C. until required for the assay. On the day of the experiment, thecells were thawed rapidly, the contents of each vial was diluted in 10mL of Iscove's modified Dulbecco's medium containing 10% fetal bovineserum (IMDM+10% FBS) and washed by centrifugation (approximately 1200r.p.m. for 10 minutes, room temperature). The supernatant was discardedand the cell pellets resuspended in a known volume of IMDM+10% FBS. Acell count (3% glacial acetic acid) and viability assessment (trypanblue exclusion test) was performed for the bone marrow sample.

Compounds

All compounds were used as solutions in DMSO at 10 mM. Two differentdosing paradigms were used depending on the study, either a six pointdose response with top concentration of 20 μM, or a three point doseresponse with a top concentration of 10 μM. In six point dose responsestudies, compounds were tested at six distinct concentrations (20, 6.7,2.2, 0.74, 0.25 and 0.082 μM), with serial dilutions prepared from the10 mM stock concentration to achieve concentrations of 6.7, 2.2, 0.74,0.25 and 0.082 mM. When added to the collagen-based media at 1:1000(v/v), the final desired test concentrations were achieved. In order togenerate the 20 μM final concentration, 8 μL of the 10 mM stock wasadded to the appropriately labeled tube. For the three point doseresponse studies, serial dilutions were prepared from the stockconcentration to achieve concentrations of 2 and 0.4 mM. When added tothe collagen-based media at 1:1000 (v/v), the final test concentrationsof 10, 2 and 0.4 μM were achieved.

Method Summary

Clonogenic progenitors of the human megakaryocytic (CFU-Mk) lineageswere set up in the media formulation described above. All compounds wereadded to the medium to give the final desired concentrations (20, 6.67.2.22, 0.74, 0.25, and 0.082 μM). Solvent control cultures (containing nocompound but 0.2% DMSO), as well as a standard control (containing nocompound and no DMSO), were also initiated.

Human megakaryocyte progenitor assays were initiated with 1.8×10⁵ cellsper culture (lot #0170525). Following 14-16 days in culture, thecultures were transferred from the 35 mm dishes to labeled glass slides,were fixed (methanol/acetone) and then stained using an anti-human CD41antibody and an alkaline phosphate detection system according tomanufacturers' instructions. The colonies were assessed microscopicallyand scored by trained personnel and divided into the followingcategories based on size; CFU-Mk (3-20), CFU-Mk (21-49), CFU-Mk (≥50).

Statistical Analyses of CFC Numbers

The mean±1 standard deviation of three replicate cultures was calculatedfor the progenitors. Two-tailed standard t-tests were performed toassess if there was a difference in the number of colonies generatedbetween solvent control and treated cultures. Due to the potentialsubjectivity of colony enumeration, a p value of less than 0.01 isdeemed significant. To calculate the concentration of 50% inhibition ofcolony growth (IC₅₀) for each compound, a dose response curve wasgenerated plotting the log of the compound concentration versus thepercentage of control colony growth using Graphpad Prism 7.

Results

Megakaryocyte colony enumeration was performed by trained personnel. Inaddition, the distribution of colony types as well as general colony andcellular morphology was analyzed. The variance in colony number detectedin replicate cultures was representative of the historical coefficientof variation for colony enumeration using these types of assays. Thenumber and distribution of colonies detected in the solvent control(0.2% DMSO) was not significantly different from the standard control(containing no compound and no DMSO). For statistical analysis colonynumbers in compound treated cultures were compared to the solventcontrol cultures. The megakaryocyte progenitor assay allows for theevaluation of more primitive progenitors, detected by the presence oflarge colonies (CFU-Mk≥50), the intermediate progenitors (CFU-Mk 21-49),detected by the presence of medium sized colonies, as well as the mostmature progenitors (CFU-Mk 3-20), detected by the presence of smallcolonies. The total CFU-Mk value is the sum of the CFU-Mk (3-20), CFU-Mk(21-49) and CFU-Mk (≥50). IC₅₀ values are determined based on the totalCFU-Mk.

Results are shown in Table 6 (IC50 shown for 6-point dose response; %control remaining @ 10 uM dose shown for 3-point dose response).

TABLE 6 % Control CFU-Mk CFU-Mk Total IC50 Total @ 10 Compound Structure(uM) uM dose 2

2 — 3

2 — 5

— 36 9

10.8 — 12

>20 — 14

— 31 17

— 28

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

The invention claimed is:
 1. A compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 3. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 4. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 5. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 6. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 7. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 8. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 9. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 10. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 11. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 12. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 13. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 14. The compound of claim1, wherein the compound is of the Formula:

or a pharmaceutically acceptable salt thereof.
 15. A compositioncomprising a compound of claim 1, or a pharmaceutically acceptable saltthereof; and a pharmaceutically acceptable carrier.
 16. A method ofinhibiting HDAC activity in a subject comprising the step ofadministering to the subject in need thereof an effective amount of acompound of claim 1, or a pharmaceutically acceptable salt thereof, orthe composition of claim 15.